The objective of this project is to develop anti-bcr-abl ribozymes (RZ) that can be used to purge stem cells in vitro of Philadelphia chromosome (Ph) positive leukemia cells from patients with PH+ acute lymphoblastic leukemia (ALL), for use in the reconstitution of Ph- hematopoiesis after myeloablative radiochemotherapy. The goal of specific aim one is first to synthesize RZs against the p190bcrabl and p210bcrabl oncogenes that can effectively cleave the mRNA of these oncogenes in Ph+ ALL cell lines and freshly isolated ALL cells. The second goal of aim one is to develop optimal strategies for the delivery and expression of the RZs in target cells, including transfection of synthetically modified RZs via liposome vectors, and transduction of Adeno-associated viral (AAV) and retroviral constructs containing the RZ genes. The third goal is to optimize the expression of the RZ gene within the viral constructs by testing a variety of promoters to drive transcription. The kinetics of the catalytic reaction of RZ and its substrate will be manipulated by exploiting naturally occurring binding proteins. After these in vitro studies are completed, the safety and efficacy of the optimal vector system found will be tested in a murine bone marrow transplant model in which transgenic mice which express the human p 190bcrabl gene develop acute leukemia a adults, and can transplant the disease to normal syngeneic mice via bone marrow cells. In specific aim two the pathogenic role of the bcr-abl oncogene in Ph+ALL will be explored by studying the impact of RZ-mediated inhibition of bcr-abl gene expression on a variety of parameters of cell growth including apoptosis. In specific aim three we plan to utilize the anti-bcr-abl RZ technology for the therapy of Ph+ ALL in humans. Our approach will be to use ablative chemotherapy and G-CSF to mobilize and collect peripheral blood stem cells (PBSC) from patients with Ph+ ALL in first or subsequent CR, and to purge those PBSC by transfecting them with RZ in liposomes and/or transducing them with the AAV/RZ or retroviral/RZ constructs. This RZ purging approach in the context of autologous BMT could offer a potentially curative therapy for those patients with Ph+ ALL for whom allogeneic BMT is not an option.